Melanosomal differentiation antigens are a family of proteins uniquely expressed on melanocytes and melanoma cells. As such, they represent rational and convenient surrogate markers to measure immune responses to melanoma in both animal model systems and human patients on clinical trials of innovative immunotherapeutic strategies. They also are very attractive targets for immunotherapies given their restricted expression on normal cells. Given the progress made during the prior funding period, we have proposed three specific aims. The first is to continue our focus on GITR (glucocorticoid-induced TNF-receptor family related protein) as a novel target of agonist immunotherapeutic strategies. We defined a novel mechanism underlying GITR-induced immune modulation with regulatory T cells (Treg) losing lineage commitment and a favorable change in the Treg:T effector cell ratio. This will be applied to a first-in-human clinical trial of an agonist monoclonal antibody to GITR, which we are currently leading as well as a Phase I trial of the Merck anti-GITR antibody. We will further these biomarker investigations by exploring the expression of immune regulatory molecules on tumor cells. We also plan to further investigate the basis for sub-optimal response at later timepoints (refractory vs. responding tumors) using assessment of these biomarkers over time. In Aim 2, we will continue our efforts to identify the most impactful and relevant combinations of immune modulators by studying the effects of dual costimulation with agonist antibodies against the TNFR superfamily receptors OX40 and GITR in transplantable and spontaneous mouse models of melanoma, using immunity to melanosomal antigens as an outcome measure. This is directly applicable to clinical trials as we have ongoing collaborations with industry partners to make clinical grade reagents available for further trials. In Aim 3, we will have a unique opportunity to directly compare two cell-based immunotherapeutic strategies, chimeric antigen receptor T cells (CAR+) and T cells bearing a transgenic T cell receptor for the same antigen that the CAR+ cells recognize (TRP1, a melanosomal antigen). We will compare different host cells as well as antigen receptors to define an optimal cell therapy to combine with the antibody combination defined in prior Aims. Our overall goal is to use immunity to melanosomal antigens to identify the most potent immunotherapeutic strategies for melanoma that can be brought into clinical trials.